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Seho Kweon,1,2 Seong Jin Park,3 Ha Kyeong Lee,1 Seo Hee Kang,4 Kwan-Young Chang,4 Jeong Uk Choi,5 Jooho Park,6 Jung-Hyun Shim,7 Jin Woo Park,7 Youngro Byun1,3 1Department of Molecular Medicine and Biopharmaceutical Science, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, 08826, Republic of Korea; 2College of Pharmacy, Chonnam National University, Gwangju, 61186, Republic of Korea; 3College of Pharmacy, Seoul National University, Seoul, 08826, Republic of Korea; 4Global R&D Center, IcureBNP, Seoul, 06170, Republic of Korea; 5College of Pharmacy, Kyung Hee University, Seoul, 02447, Republic of Korea; 6Department of Biomedical & Health Science, Konkuk University, Chungju, 27478, Republic of Korea; 7College of Pharmacy and Biomedical and Healthcare Research Institute, Mokpo National University, Jeonnam, 58554, Republic of KoreaCorrespondence: Jin Woo Park; Youngro Byun, Tel +82 61 450 2704, Email jwpark@mokpo.ac.kr; yrbyun@snu.ac.krBackground: For maintenance therapy in type 2 diabetes, glucagon-like peptide-1 agonist (GLP-1A), which exhibits low cardiovascular risk and high efficacy, is a promising peptide therapeutic. However, developing an oral GLP-1A presents challenges due to the analog’s poor cellular permeability and gastrointestinal (GI) stability.Methods: To mitigate such limitations, an oral nanoformulation of liraglutide (LG) was designed and achieved by combining LG with bile acid derivatives using the nanoprecipitation method. This strategy allowed the bile acid moieties to localize at the nanoparticle surface, enhancing the binding affinity for apical sodium-dependent bile acid transporter (ASBT) and improving GI stability. The in vitro characteristics, cellular permeability, and absorption mechanisms of the LG nanoformulation (LG/TD-NF) were thoroughly investigated. Furthermore, the in vivo oral absorption in rats and the glucose-lowering effects in a diabetic (db/db) mouse model were evaluated.Results: The LG/TD-NF produced neutral nanoparticles with a diameter of 58.7 ± 4.3 nm and a zeta potential of 4.9 ± 0.4 mV. Notably, when exposed to simulated gastric fluid, 65.7 ± 3.6% of the LG/TD-NF remained stable over 120 min, while free LG was fully degraded. Relative to unformulated LG, the Caco-2 cellular permeability of the nanoformulation improved, measuring 10.9 ± 2.1 (× 10− 6 cm/s). The absorption mechanism prominently featured endocytosis simultaneously mediated by both ASBT and epidermal growth factor receptor (EGFR). The oral bioavailability of the LG/TD-NF was determined to be 3.62% at a dosage of 10 mg/kg, which is 45.3 times greater than that of free LG. In a diabetes model, LG/TD-NF at 10 mg/kg/day exhibited commendable glucose sensitivity and reduced HbA1c levels by 4.13% within 28 days, similar to that of subcutaneously administered LG at a dosage of 0.1 mg/kg/day.Conclusion: The oral LG/TD-NF promotes ASBT/EGFR-mediated transcytosis and assures cellular permeability within the GI tract. This method holds promise for the development of oral GLP-1A peptides as an alternative to injections, potentially enhancing patient adherence to maintenance therapy.Keywords: nanoformulation of GLP-1A, oral liraglutide, ASBT-mediated endocytosis, EGFR

Abstract Background Despite the effectiveness of glucagon-like peptide-1 agonist (GLP-1A) in the treatment of diabetes, its large molecular weight and high hydrophilicity result in poor cellular permeability, thus limiting its oral bioavailability. To address this, we developed a chimeric GLP-1A that targets transporter-mediated endocytosis to enhance cellular permeability to GLP-1A by utilizing the transporters available in the intestine, particularly the apical sodium-dependent bile acid transporter (ASBT). Methods In silico molecular docking and molecular dynamics simulations were used to investigate the binding interactions of mono-, bis-, and tetra-deoxycholic acid (DOCA) (monoDOCA, bisDOCA, and tetraDOCA) with ASBT. After synthesizing the chimeric GLP-1A-conjugated oligomeric DOCAs (mD-G1A, bD-G1A, and tD-G1A) using a maleimide reaction, in vitro cellular permeability and insulinotropic effects were assessed. Furthermore, in vivo oral absorption in rats and hypoglycemic effect on diabetic db/db mice model were evaluated. Results In silico results showed that tetraDOCA had the lowest interaction energy, indicating high binding affinity to ASBT. Insulinotropic effects of GLP-1A-conjugated oligomeric DOCAs were not different from those of GLP-1A-Cys or exenatide. Moreover, bD-G1A and tD-G1A exhibited improved in vitro Caco-2 cellular permeability and showed higher in vivo bioavailability (7.58% and 8.63%) after oral administration. Regarding hypoglycemic effects on db/db mice, tD-G1A (50 μg/kg) lowered the glucose level more than bD-G1A (50 μg/kg) compared with the control (35.5% vs. 26.4%). Conclusion GLP-1A was conjugated with oligomeric DOCAs, and the resulting chimeric compound showed the potential not only for glucagon-like peptide-1 receptor agonist activity but also for oral delivery. These findings suggest that oligomeric DOCAs can be used as effective carriers for oral delivery of GLP-1A, offering a promising solution for enhancing its oral bioavailability and improving diabetes treatment. Graphical Abstract